Involved in Vegetal mRNA Localization in Xenopus Oocytes
Dissertation
zur Erlangung des mathematisch-naturwissenschaftlichen Doktorgrades
"Doctor rerum naturalium"
der Georg-August-Universität Göttingen
vorgelegt von
Patrick Kobina Arthur
aus
Saltpond, Ghana.
Göttingen, 2008.
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D 7
Referent: Prof. Dr. Tomas Pieler
Korreferent: Prof. Dr. Ernst A. Wimmer Date of Thesis Submission: 26th May, 2008.
Date of Thesis Disputation: 27th June, 2008
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Herewith I declare, that I prepared the Doctoral thesis
“Identification and Functional Characterization of Trans-acting Factors Involved in Vegetal mRNA Localization in Xenopus Oocytes”
on my own and with no other sources and aids than quoted.
Patrick Kobina Arthur ...
Date of submission ...
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… . to Marian my wife with love.
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Now to the King eternal, immortal, invisible, the only God, be honor and glory forever and ever – (I Timothy 1:17 - NIV)
I wish to express my heartfelt appreciation to Prof. Dr. Tomas Pieler for offering me the opportunity to do my PhD thesis project in his laboratory and for the continuous support and encouragement that has seen me thus far. I do hope to carry on with what I have learnt from my time here to benefit others and to make you proud for the good work invested in me. I thank Prof. Wimmer for accepting to be my korreferent. The efforts of Frau Manuela Manafas towards solving the numerous administrative difficulties I got into cannot go unrecognized; she offered such timely and effective assistance that made me lost for words to express my gratitude.
My entire family has supported me in such a manner that made it possible to stay the course and to take this pursuit to a good end, my mother Felicia and father Joseph Arthur, spoke to situations with such wisdom and care it made possible not to spare any effort at reaching my set goals. The last mile, it is said, is always the longest mile and I could not have made it through it all without the love and tacit support of my wife Marian; I am grateful for the constant warning that if I did not complete, I should not consider it an option to return home.
I am grateful to all the members of the department who contributed in various ways to make my work possible, especially the “transport group” – Jana Löber, Katsiaryna Tarbashevich, Stefanie Oswald, Katja Koebernick and Maike Claussen; it was also a joy working with former group members – Britta Dreier*, Susanne Koch, Ines Eckhardt, Marc Püschel, and Katja Horvay.
Working in the department was also made a particularly pleasant experience by some very wonderful people namely Andreas Nolte, Marion Dornwell, Ilona Wunderlich and Gudrun Kracht.
Special thanks to Christine Jäckh for her kind support all the time and also to Dr. Yonglong Chen and Dr. Jacob Souopgui for taking a special interest in my work. I appreciate contributions made by my friends outside the lab – Dr. Henry Acquah* and Edward Onumah*; as well as all my compatriots in Göttingen. *Thanks for proofreading parts of the manuscript.
Many thanks to Dr Olaf Jahn, MPI-em, Göttingen for the great collaboration on protein identification part of my work, and also for going the extra step offering advice and ideas that helped me great deal. Finally, I can state that “I have seen thus far not because I am a giant but I have stood on the shoulders of giants”. My deepest gratitude to all, who could not be mentioned, but have been a giant for me in my advance towards the PhD.
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TABLE OF CONTENTS
Title Page ...……….i
Thesis advisers………...ii
Affidavit………..iii
Dedication……….…iv
Acknowledgements……….……….………..v
1. INTRODUCTION ... 10
1.1. Biological importance of mRNA localization in different systems... 10
1.2. Oogenesis in Xenopus laevis ... 14
1.3. Pathways of vegetal mRNA localization... 17
1.3.1. Early (METRO) pathway ... 17
1.3.2. Late pathway ... 18
1.3.3. Intermediate pathway... 19
1.4. Mechanisms of Vegetal mRNA localization ... 20
1.4.1. Diffusion and entrapment ... 21
1.4.2. Active transport ... 22
1.4.3. Cis-acting elements... 25
1.4.4. Trans-acting factors ... 26
1.4.4.1. Vg1 mRNA binding protein - Vg1RBP... 26
1.4.4.2. XStaufen1 protein ... 27
1.4.4.3. VgRBP60/PTB protein ... 28
1.4.4.4. Xenopus 40LoVe protein... 29
1.4.4.5. Xenopus Proline-rich RNA binding protein - Prrp... 29
1.4.4.6. VgRBP71 protein... 30
1.5. Transport RNP: size, composition and structure... 30
1.6. Translational control of localized mRNAs... 31
1.6.1. Drosophila oocytes/embryos ... 32
1.6.2. Neurons and Fibroblast cells ... 32
1.6.3. Xenopus oocytes... 32
1.7. Aims of the project ... 34
2. MATERIALS AND METHODS ... 35
2.1.1. Model Organism ... 35
2.1.2. Bacteria strains... 35
2.1.3. List of Chemicals... 35
2.2. DNA methods ...36
2.2.1. Plasmid constructs and protein production... 36
2.2.2. Polymerase Chain Reaction (PCR) ... 37
2.2.3. Site-directed Mutagenesis ... 38
2.2.4. Reverse Transcription-PCR (RT-PCR)... 40
2.2.5. Quantitative RT-PCR (qPCR) ... 42
2.2.6. Restriction Endonuclease Digestion... 43
2.2.7. Ligation... 43
2.2.8. Preparation of Chemical Competent cells ... 44
2.2.9. Preparation of Electrocompetent Cells ... 44
2.2.10. Transformation of chemical competent cells... 45
2.2.11. Transformation by Electroporation ... 45
2.2.12. Plasmid Prep... 46
2.2.13. Agarose Gel Electrophoresis... 46
2.3. RNA methods ...46
2.3.1. In vitro Transcription... 46
2.3.2. Digoxigenin Labelled RNA... 47
2.3.3. Alexa labelled RNA... 48
2.3.4. Radiolabelled RNA ... 48
2.3.5. Total RNA Isolation Using Trizol Reagent ... 48
2.3.6. RNAeasy Cleanup... 49
2.3.7. RNA Precipitation Techniques ... 49
2.3.7.1. Isopropanol Precipitation ... 49
2.3.7.2. Sodium Acetate/Ethanol Precipitation ... 49
2.3.7.3. Ethanol Precipitation ... 50
2.3.8. Whole Mount In situ Hybridization (WMISH)... 50
2.3.8.1. Formaldehyde Fixation ... 50
2.3.8.2. Rehydration ... 50
2.3.8.3. Proteinase K treatment ... 51
2.3.8.4. Hybridization ... 51
2.3.8.5. Washing of Unhybridized Probe ... 51
2.3.8.6. Antibody Incubation ... 52
2.3.8.7. Color Reaction ... 52
2.3.9. RNase Protection Assay ... 53
2.3.10. Urea-PAGE electrophoresis ... 54
2.4. Oocyte Manipulations... 55
2.4.1. Oocytes Isolation... 55
2.4.2. Microinjection into oocytes ... 55
2.4.2.1. Localization Assay... 55
2.4.2.2. Nucleo-cytoplasmic Transport Assay ... 56
2.4.2.3. MS2-Tethering Assay ... 57
2.4.3. Oocyte Extract Preparation ... 58
2.4.4. Preparation of Vitellogenin... 58
2.5. Protein Methods...59
2.5.1. Bradford Assay... 59
2.5.2. Protein Precipitation Techniques ... 59
2.5.2.1. Acetone... 59
2.5.2.2. NaDOC-TCA Acetone ... 59
2.5.2.3. Phenol/Chloroform Extraction... 60
2.5.3. SDS-PAGE Electrophoresis... 60
2.5.4. Silver Staining... 61
2.5.5. Colloidal Coomassie Blue Staining ... 61
2.5.6. Western Blotting... 62
2.5.7. Two Dimensional Gel Electrophoresis... 63
2.5.8. Mass Spectroscopic Protein Identification... 64
2.5.9. Glycerol Gradient Centrifugation and Biochemical Enrichment ... 64
2.5.10. Anion Exchange Chromatography ... 65
2.5.11. Immunoprecipitation... 65
2.5.12. UV crosslinking Assay ... 66
2.5.13. Electrophoretic Mobility Shift Assay (EMSA)... 67
2.5.14. Paraffin Embedding and Sectioning ... 68
2.5.15. Immunostaining on Oocyte sections ... 68
2.6. Embryo Injections... 70
3. RESULTS ... 71
3.1. Identification Xenopus ElrA and ElrB as vegetal localization element binding proteins ... 71
3.2. ElrB isoforms and ElrA exhibit similar specificities in binding to vegetal localization elements ... 78
3.3. ElrA/B proteins are part of one RNP complex together with Vg1RBP, XStaufen1, VgRBP60 and 40LoVe... 84
3.4. Inhibition of ElrA/B binding correlates with a loss of vegetal mRNA transport ... 92
3.5. Effect of Elr-type proteins on mRNA stability and translation in oocytes... 97
3.6. Loss of ElrA/B binding strongly decreases mRNA stability in primordial germ cells in embryos ... 101
3.7. Subcellular distribution of Elr-type proteins in Xenopus ... 103
3.8. ElrA/B proteins are constitutive components of RNPs throughout oogenesis ... 108
4. DISCUSSION ... 111
4.1. Elav... 111
4.2. Vegetal localization proteins are components of a large RNP ...112
4.3. Isolation of ElrA/B as a novel vegetal localization element binding proteins ... 113
4.4. Heterogeneity of the localizing RNP ... 114
4.5. Identity of protein components of ElrA/B RNP complexes ... 117
4.6. Participation of Elr-type proteins in vegetal mRNA localization ... 121
4.7. ElrA/B and translational control in oocytes ... 122
4.8. ElrA/B and mRNA stability in oocytes ... 124
4.9. Mutations inhibiting ElrA/B binding lead to a reduction in mRNA stability in PGCs 125 4.10. Subcellular distribution of ElrA/B, a constitutive RNP component throughout oogenesis ... 126
5. SUMMARY ... 127
6. LITERATURE REFERENCES... 128
7. APPENDIX ... 148
7.1. Appendix I... 148
7.1.1.Appendix I: Density gradient profile of localization proteins and ribosomes (leg)..148
7.1.2.Appendix I: Density gradient profile of localization proteins and ribosomes (Fig). 149 7.2. Appendix II... 150
7.2.1. Appendix II: Identification of proteins in the RNP S200 – pH3-10(Legend)... 150
7.2.2. Appendix II: Identification of proteins in the RNP S200 – pH3-10(Gel)... 151
7.2.3. Appendix II: Identification of proteins in the RNP S200 – pH3-10(Table)... 152
7.3. Appendix III... 153
7.3.1. Appendix III: Identification of proteins in the RNP S200 – pH7-11(Legend)... 153
7.3.2. Appendix III: Identification of proteins in the RNP S200 – pH7-11(Gel)... 154
7.3.3. Appendix III: Identification of proteins in the RNP S200 – pH7-11(Table)... 155
7.4. Appendix IV ... 156
7.4.1. Appendix IV: Identification of proteins in the HS RNP S200 – pH3-10(Legend)... 156
7.4.2. Appendix IV: Identification of proteins in the HS RNPS200 – pH3-10(Gel)... 157
7.4.3. Appendix IV: Identification of proteins in the HS RNPS200 – pH3-10(Table)... 158
7.4.4. Appendix IV: Identification of proteins in the HS RNP S200 – pH3-10(Table-contd)159 7.5. Appendix V ... 160
7.5.1. Appendix V: Identification of proteins in the HS RNPS200 #2 – pH3-10(Legend).. 160
7.5.2. Appendix V: Identification of proteins in the HS RNPS200 #2 – pH3-10(Gel)... 161
7.5.3. Appendix V: Identification of proteins in the HS RNPS200 #2 – pH3-10(Table)... 162
7.6. Appendix VI ... 163
7.6.1. Appendix VI: Identification of proteins associated with 40LoVe –pH3-10(Legend)163 7.6.2. Appendix VI: Identification of proteins associated with 40LoVe –pH3-10(Gel)……164
7.6.3. Appendix VI: Identification of proteins associated with 40LoVe –pH3-10(Table).. 165
7.7. Appendix VII: Identification of proteins associated with ElrA/B –pH3-10 (Gel)... 166
7.8. Appendix VIII: Identification of proteins associated with 40LoVe –pH3-10 (Gel)... 167
Curriculum vitae………168
List of Tables
Table 1.1 Early localized mRNA in Xenopus oocytes... 18
Table 1.2 mRNAs localized through late pathway... 19
Table 1.3 mRNAs localized via the intermediate pathway.... 19
Table 2.1 XDE LE mutagenesis primers... 39
Table 3.1 List of proteins identified from RNP S200 with predicted functional link to RNA.. 76
Table 3.2 List of proteins co-precipitating with the Elr-type proteins... 89
List of Figures
Figure 1.1 Roles of mRNA localization in a variety of systems [from (Kloc et al., 2002b)]... 12
Figure 1.2 Examples of mRNA localization in different biological systems... 14
Figure 1.3 Stages of Xenopus laevis oogenesis, [modified from (Horvay, 2005)]... 15
Figure 1.4 Section of stage I-VI oocytes [from (Hausen and Riebesell, 1991)]... 16
Figure 1.5 Annotation of oocyte components in a section of a stage VI oocyte,... 17
Figure 1.6 Pathways of vegetal mRNA localization in oocytes... 20
Figure 1.7 Model of the mechanism of diffusion and entrapment used by early localized mRNAs in oocytes... 22
Figure 1.8 Model of the active transport mechanism used by mRNAs localizing via the late pathway... 24
Figure 1.9 Structure of mRNA depicting its vegetal localization element in the 3´ UTR... 25
Figure 1.10 Domain structure of Xenopus Vg1RBP protein... 26
Figure 1.11 Domain structure of Xenopus Staufen 1 protein... 28
Figure 1.12 Model for the function of the Drosophila Staufen protein... 28
Figure 1.13 Domain structure of Xenopus VgRBP60 protein, depicting the 4 RRM domains29 Figure 1.14 Domain structure of Xenopus 40LoVe (Localization Vegetal) protein depicting the two RRM domains.... 29
Figure 1.15 Domain structure of Xenopus Prrp protein depicting the 2 N-terminal RRM domains and a C-terminal Proline-rich domain... 30
Figure 1.16 Domain structure of Xenopus VgRBP71 protein, showing 4 KH domains drawn to the scale as predicted by the NCBI-protein blast program... 30
Figure 2.1 Scheme for PCR-based Multiple Site directed Mutagenesis... 39
Figure 3.1 Biochemical enrichment of vegetal localization element binding proteins... 74
Figure 3.2 Identification of RNA binding proteins contained in RNPS200... 75
Figure 3.3 Identification of ElrA/B protein as vegetal localization element binding proteins77 Figure 3.4 Anion exchange chromatographic (ANX) profile of RNA binding proteins in RNP S200... 79
Figure 3.5 Vg1RBP and Elr-type proteins bind vegetal localization elements with different specificities... 81
Figure 3.6 Comparison of domain structure and sequences of putative Elr-type isoforms
expressed in the oocyte... 82
Figure 3.7 Vegetal localization element binding properties are indistinguishable for the tagged versions of and the putative endogenous ElrB isoforms in the oocyte... 83
Figure 3.8 ElrA and B proteins Interact with Vg1RBP and XStaufen1 in an RNA-dependent manner... 85
Figure 3.9 ElrA/B interact with localization proteins VgRBP60 and 40LoVe in an RNA dependent manner... 85
Figure 3.10b Identification of protein components of immuno-isolated ElrA/B RNPs (analytical scale)... 87
Figure 3.11 Identification of protein components of immuno-isolated ElrA/B RNPs... 90
Figure 3.12 Novel RAP55 related protein co-immunoprecipitated with ElrA/B proteins... 92
Figure 3.13 Mutagenesis of putative ElrA/B binding sites in XDE-LE... 93
Figure 3.14 Interference with ElrA/B-binding by mutagenesis blocks vegetal localization of the XDE LE... 94
Figure 3.15 Antisense morpholino mediated Interference with ElrA/B-binding blocks vegetal localization of the XDE-LE... 95
Figure 3.16 Inhibition of vegetal RNA localization by antisense morpholino oligonucleotides blocking ElrA/B binding to XDE LE... 97
Figure 3.17 ElrA/B proteins moderately upregulate translation in oocytes and loss of binding mutation in XDE LE does not markedly alters translation levels of reporter mRNA... 98
Figure 3.18 Interference with ElrA/B binding to XDE LE does not influence mRNA stability in oocytes... 100
Figure 3.19 Loss of binding mutation in XDE LE strongly decreases mRNA stability in PGCs during embryogenesis.... 102
Figure 3.20a Subcellular distribution of ElrA and B in stage III oocytes... 105
Figure 3.20b Subcellular distribution of ElrA and B in stage IV oocytes... 106
Figure 3.22 ElrA/B and Vg1RBP proteins are constitutive RNP components throughout oogenesis... 109
Figure 3.23 Profile of total protein from stage I oocytes on glycerol gradient... 110
Figure 4.1 Domain structure of Xenopus Elav protein, showing 3 RRM domains... 112 Figure 4.2 Model of RNP heterogeneity exhibited by the various vegetal localization
elements... 116
1. Introduction
mRNA localization is a multi-step process of transporting mRNA molecules to a specific domain of a cell; it is initiated by the recognition of a cis-acting signal by trans-acting factors and this most often ultimately results in local protein synthesis and function. It is a mechanism used in wide a variety of biological processes for the establishment of asymmetry vital for cellular function or embryonic development. The occurrence of this process has been reported in several biological systems such as Drosophila oocyte, yeast, Xenopus oocyte, fibroblasts and neuronal cells (Dahm and Kiebler, 2005; Gonsalvez et al., 2005; Kloc et al., 2001; Kloc et al., 2002b; St Johnston, 2005).
1.1. Biological importance of mRNA localization in different systems
In Drosophila, the two prominent examples of mRNAs of Bicoid and Oskar are transcribed in the nurse cells and localize to anterior (Fig. 1.2A) and posterior (Fig.
1.2B) poles of the oocyte respectively. The bicoid protein thus forms an anterior- posterior morphogen gradient (illustrated in Fig. 1.1B), that organizes the anterior structures of the embryo (Driever and Nusslein-Volhard, 1988a; Driever and Nusslein-Volhard, 1988b; Driever et al., 1990). Oskar protein on the other hand serves a dual developmental function of the determination of germ cell fate and posterior polarity (Lehmann and Ephrussi, 1994). In addition Oskar mRNA plays a translation-independent role during oogenesis perhaps as scaffold or regulatory RNA, since Oskar mRNA null mutants were found to be sterile due to arrest in early oogenesis (Jenny et al., 2006). This defines an exception to the notion that localized mRNAs exert their function solely by ensuring high local protein concentration. In Xenopus oocytes, examples of translation-independent function for localized mRNAs have also been described for VegT and Xlsirt mRNAs, which are required for the cytokeratin mediated anchoring of vegetally localized mRNAs at the cortex (Kloc et al., 2005).
Another instance for the biological relevance of mRNA localization is the translation of ß-actin mRNA localized at the leading edge of fibroblast cell, (Fig 1.1A and 1.2E); this then ensures that actin polymerization is in the leading edge of the cell to enable directional migration to occur. The same is true for ß-actin
mRNA in neurons (Fig 1.1E), where the same mechanism contributes to the regulation of growth and guidance of axons (Dahm and Kiebler, 2005). The mRNA encoding myelin basic protein (MBP) is similarly localized in oligodendrocytes to the dendrites, this is meant to prevent translation of this highly positively charged protein anywhere in the cell except for the site of myelination (Smith, 2004). In budding yeast (Fig 1.2 C), mating type switching is regulated by the exclusive translation of localized ASH1 mRNA in the daughter cell (Chartrand et al., 2001;
Gonsalvez et al., 2005). The Ash1 protein in the daughter cell functions as a transcriptional factor which inhibits the expression of the endonuclease HO, which promotes the mating-type switching process (Tekotte and Davis, 2002).
The mRNA localization process is also used to restrict certain transcripts to subcellular structures, e.g. cyclin B1 mRNA is targeted to mitotic spindles in Xenopus embryos (Groisman et al., 2000). This local translation of cyclin B1 at the spindle apparatus is believed to regulate the cell cycle. There are now several mRNAs identified that exhibit localization to the perinuclear region as well as many other subcellular domains previously not described (Lecuyer et al., 2007).
These further indicate the importance mRNA localization as an important mechanism for the fine tuning of gene expression and as such aberration in this process could lead to defects in cell function, the developmental program of whole organisms and it can also result in disease (Bassell and Kelic, 2004; Wang et al., 2007) .
During Xenopus oogenesis, the process of vegetal mRNA localization contributes to laying the foundation for germ layer formation and germ cell development later during embryogenesis. There are two distinct populations of localized mRNA in Xenopus oocytes; the first group is involved in germ layer determination, the best studied examples are Vg1 (Melton, 1987; Rebagliati et al., 1985) and VegT mRNA (Horb and Thomsen, 1997; Stennard et al., 1996). These mRNAs encode a TGF beta homolog (Melton et al., 1989) and a T-box transcription factor (Stennard et al., 1996), respectively. They are inherited largely by the vegetal-most blastomeres resulting in vegetal-animal protein gradients essential for the induction of endodermal and mesodermal germ layers (Kofron et al., 1999; Zhang et al., 1998). The second group of mRNAs, such as Xpat, Xcat2, and Xdazl, is
involved in the specification of germ cells (Houston et al., 1998; Hudson and Woodland, 1998; Mosquera et al., 1993).
Figure 1.1 Roles of mRNA localization in a variety of systems [from (Kloc et al., 2002b)]
(A) A High level of a protein is produced in a specific domain of a cell as illustrated by ß-actin mRNA and protein accumulation at the leading edge of a fibroblast. (B) The generation of morphogen gradients in oocytes and embryos as found in Drosophila; bicoid mRNA localization produces a gradient of Bicoid protein. (C) Cell lineage is specified by localized mRNAs where determinants are partitioned unequally into daughter cells; Xenopus germplasm containing early localized mRNA specify PGCs. (D) mRNAs associate with different organelles or cellular structures, as exemplified by the localization of cyclin B mRNA at the mitotic spindle poles. (E) Some mRNAs are localized to a specific region of a cell to allow for local translation, e.g. mRNAs at the synapses of neurons (red).
Figure 1.2 Examples of mRNA localization in different biological systems
(A) Drosophila Bicoid mRNA is localized to the anterior pole of a freshly laid egg [from (Irion and St Johnston, 2007)]. (B) Drosophila Oskar mRNA is localized to the posterior pole of a freshly laid egg [from (Vanzo and Ephrussi, 2002)]. (C) ASH1 mRNA is localized to the distal tip of anaphase cells, (D) the corresponding DAPI (blue)/Nomarski merged image [from (Gonsalvez et al., 2005)].
(E) ß-actin mRNA (red) localizes to the fibroblast's leading edge, -actin protein is shown in green, and the nucleus is stained in blue [from (Dahm and Kiebler, 2005)] (F) ß-actin mRNA localization in the neurite and growth cone; (G) ß-actin protein is highly enriched in the growth cone and filopodia [from (Kloc et al., 2002b)].
1.2. Oogenesis in Xenopus laevis
Oogenesis in Xenopus laevis is divided into six stages (Dumont, 1972); the oogonium is specified from a self-renewing stem cell which gives rise to a large number of oocytes in the female. During oogenesis, the oocyte which is arrested in diplotene stage of prophase I, increases dramatically in size and accumulates all the material needed for the early stages of embryo development. There are two major phases; the previtellogenesis (stage I) and the vitellogenesis phase. At stage II, the oocyte begins to take up large amounts of vitellogenin from the blood by micropinocytosis. Vitellogenin is a 470 kDa protein produced in the liver of the female frog and is delivered to the oocytes through the bloodstream; it serves as the source of nutrition for the embryo at the early stages of development (Gilbert, 2000).
The stage I oocyte is transparent, with a centrally placed nucleus; the first sign of asymmetry is the attachment of the mitochondria cloud (also called the Balbiani body) to one side of the nucleus facing the future vegetal hemisphere (Fig. 1.3 and 1.4A) (Guraya, 1979). The Balbiani body consists of a number of mitochondria, endoplasmic reticulum, germplasm and several mRNAs mostly implicated in the process of germ line specification. A sub-structure of the Balbiani body pointing to the vegetal pole is described as message transport organiser (METRO), it contains the germinal granules. The stage II oocyte begins to acquire pigmentation as well as to take up vitellogenin, whilst the mitochondrial cloud break down into islands and move towards the vegetal cortex. In stage III – VI there is a sharp contrast of the highly pigmented animal half with the vegetal half remaining lightly pigmented (Fig 1.3), the nucleus starts to shift to the animal half
from stage IV on becoming clearly evident in stage VI (Fig 1.4F), the annotated ultra-structure of the fully grown stage VI oocyte is shown in (Fig. 1.5). Although the oocyte appears radially symmetrical, there are certain mRNA molecules which localize either to the animal or to the vegetal pole. Some of these mRNAs localizing to the vegetal cortex encode proteins which have been shown to function in embryo axis specification and germ cell development, as mentioned above (Kloc et al., 2001).
Figure 1.3 Stages of Xenopus laevis oogenesis, [modified from (Horvay, 2005)]
Stage I oocytes (50-300 µm in diameter) are transparent with a centrally placed nucleus. Stage II oocytes are 300-450 µm in diameter and opaque. Stage III oocytes (450-600 µm) are lightly and uniformly pigmented. Stage IV oocytes (600-1000 µm) have the nucleus placed towards the pigmented animal hemisphere. Stage V oocytes are 1000-1100 µm, and Stage VI oocytes are fully-grown with a diameter of 1100-1300 µm.
Maturation of oocytes into eggs is triggered by progesterone which is secreted from the follicle cells, leading to the resumption and completion of meiosis. The response of the late stage VI oocytes to the hormone is indicated by germinal vesicle break-down, which appears within 6 hours of stimulation (Gilbert, 2000).
The oocyte which has been arrested in the diplotene stage of prophase I resumes and completes the process of meiosis. There are several post-transcriptional events that take place during maturation, transcription in general seizes completely in late stage VI, and several stored maternal mRNA become translationally active. This stimulation of translation of the quiescent mRNAs, including c-mos, cyclin mRNAs, is correlated strongly to the extension of their poly A tails as well as increased methylation of the 5´ cap structure (Barkoff et al., 1998; Gillian-Daniel et al., 1998; Sheets et al., 1994; Sheets et al., 1995).
Figure 1.4 Section of stage I-VI oocytes [from (Hausen and Riebesell, 1991)]
(A) Late stage I oocyte with the nucleus showing a slight depression at the side of Balbiani body attachment, displacing the nucleoli. A dense layer of follicle cells covers the oocyte. (B) Early stage II oocyte showing mitochondrial cloud fragments, parts of which surround the nucleus whilst the other part is transported towards the future vegetal pole. (C) Late stage II oocyte, Balbiani body completely fragmented, with part of it settled at the vegetal cortex. (D) Stage III oocyte, Balbiani body almost completely localized to the vegetal cortex, yolk platelets are uniform in size and positioned outwardly in the cytoplasm, cortical granules line the plasma membrane. (E) Stage IV oocytes, the pigment granules make the animal cortex thicker than the vegetal one. The yolk platelets in the vegetal half are larger than at the animal half. (F) Stage VI oocyte, the nucleus is clearly placed in the animal half, pigment granules appear as a dark layer beneath the plasma membrane.
Figure 1.5 Annotation of oocyte components in a section of a stage VI oocyte, [from (Hausen and Riebesell, 1991)]
1.3. Pathways of vegetal mRNA localization
Localization of mRNA to the vegetal cortex of oocytes occurs via two main early and late pathways and a third one that combines features of the first two and is therefore referred to as the intermediate pathway. The mRNAs transported via the early pathway are implicated mainly in germ cell specification whilst those using the late pathway are linked to germ layer specification. There is however an interesting exception in XDead end mRNA which is localized by the late pathway but only becomes restricted to the germplasm and PGCs during early embryogenesis (Horvay et al., 2006).
1.3.1. Early (METRO) pathway
There are several examples of mRNAs that use this pathway in localizing to the vegetal cortex; some of these are listed below (Table 1.1). These mRNAs are first enriched in the METRO sub-region of the mitochondrial cloud during stage I of oogenesis by diffusion and entrapment within this structure. Localization to the vegetal cortex occurs passively by stage II of oogenesis, when the Balbiani body disintegrates and migrates to the vegetal cortex (Kloc et al., 2001). At the cortex
these mRNAs characteristically occupy a narrow region, as shown in Fig. 1.6.
Early localized mRNAs remain associated with the germplasm at the vegetal cortex throughout oogenesis and early embryogenesis, where they are selectively taken up by some vegetal blastomeres which later give rise to primordial germ cells (Hudson and Woodland, 1998). There are exceptions to the general rule for early localized mRNAs to encode for proteins that function in germ cell development, e.g. Xwnt11 is reported to be involved in axis specification (Ku and Melton, 1993).
Table 1.1 Early localized mRNA in Xenopus oocytes
Localized mRNA Encoded protein function References
Xcat2 Zn finger protein, related to Nanos (Mosquera et al., 1993) Xdazl RNA binding protein, related to DAZ (Houston et al., 1998)
Xpat Novel protein (Hudson and Woodland,
1998)
Xwnt11 Wnt ligand (Ku and Melton, 1993)
Xlsirts Non-coding RNA (Kloc et al., 1993)
XNIF Novel protein (Claussen et al., 2004)
XGRIP2.1
Glutamate receptor interacting protein
(Tarbashevich et al., 2007)
Centroid DEAD-box RNA Helicase (Kloc and Chan, 2007)
1.3.2. Late pathway
mRNAs transported by the late pathway are found to be uniformly distributed in the cytoplasm of the stage I-II oocyte excluding the Balbiani body (Melton, 1987);
several of them are also absent from the germplasm in embryos (Kloc et al., 1998). These mRNAs become enriched within a wedge-shaped structure in the cytoplasm between the nucleus and the vegetal cortex, which overlaps with a sub- domain of the ER in late stage II oocytes (Deshler et al., 1997). During mid oogenesis, mRNAs gets localized by an active transport mechanism to the vegetal cortex, where they become tightly anchored. Characteristically, these mRNAs occupy the entire vegetal cortex, which is one of the major distinctions from the early pathway. It has been observed that early localized mRNAs, when injected into stage III-IV oocytes, are able to use the late pathway of transport to the vegetal cortex as well. Vg1 mRNA, as well as other late localized mRNAs remain anchored to the vegetal cortex throughout oogenesis, but are released upon
oocyte maturation (Thomsen and Melton, 1993). Examples of mRNAs localized via the late pathway are listed in the Table 1.2 (below)
Table 1.2 mRNAs localized through late pathway
Localized mRNA Encoded protein function References
Vg1 TGF-ß family member (Rebagliati et al., 1985) VegT Transcription factor (Stennard et al., 1996) XDead end RNA binding protein (RRM domain) (Horvay et al., 2006)
XPTB Phosphotyrosine binding (Claussen, 2002)
Xvelo1 Novel protein (Claussen and Pieler, 2004)
Velo7 Metabolic enzyme (Horvay, 2005)
Velo76 Novel protein (Horvay, 2005)
1.3.3. Intermediate pathway
This pathway combines features of both early and late pathways, in that mRNAs, e.g. Velo45 mRNA (Fig 1.6), transported by this pathway are uniformly distributed throughout the cytoplasm in stage I oocytes, excluded from the Balbiani body like is the case for late pathway. In stage II oocyte, these mRNAs accumulate in the Balbiani body in a pattern similar to the early pathway, then in stage III enrich in the wedge-shape structure, finally localizing broadly at the vegetal cortex reminiscent of the late pathway (Kloc et al., 2001). What is not clearly understood is whether there exists a distinct intermediate transport pathway, or if these mRNAs are able to use both pathways depending on which is operational at the time of their export into the cytoplasm (King et al., 2005). Examples of mRNAs localizing via this pathway are listed below in Table 1.3.
Table 1.3 mRNAs localized via the intermediate pathway.
Localized mRNA Encoded protein function References
fatvg Adipophilin (Chan et al., 1999)
Hermes RNA binding protein (RRM domain) (Zearfoss et al., 2004) Xotx1 Transcription factor (Pannese et al., 2000)
Velo40 Protein kinase (Horvay, 2005)
Velo45 Putative Ubiquitin ligase (Dreier, 2005)
Figure 1.6 Pathways of vegetal mRNA localization in oocytes
In situ hybridization detection of mRNAs in stage I-VI localized via the three different pathways to the vegetal cortex of Xenopus oocytes. Xpat mRNA is early localized (pictures by the courtesy of Katja Koebernick), Velo45 mRNA is localized via the intermediate pathway [picture taken from (Dreier, 2005)], and XPTB mRNA is late localized (pictures by the courtesy of Katja Koebernick).
1.4. Mechanisms of Vegetal mRNA localization
Four distinct mechanisms are known to be used by a variety of systems to achieve mRNA localization to a distinct region of the cell; vegetal mRNA transport occurs in Xenopus oocytes via two of these mechanisms. Firstly, there is a diffusion and local entrapment mechanism; in the Drosophila oocyte, the posterior pole localization of Nanos mRNA and proteins such as Oskar, Tudor and Vasa in
the pole plasm are required for RNA anchoring (Forrest and Gavis, 2003; Wang et al., 1994). A similar situation holds true for early localized mRNAs in Xenopus oocytes (Chang et al., 2004).
The second mechanism is local RNA stabilization; the level of Hsp83 mRNA is found to be markedly reduced within the developing Drosophila embryo, except at the posterior pole, where pole plasm components are thought to mediate its stabilization (Bashirullah et al., 1999). Thirdly, local mRNA synthesis is known to be responsible for the localization of mRNAs encoding different subunits of the acetylcholine receptor in multinucleate myofibers (Merlie and Sanes, 1985).
The fourth and perhaps most widely used mechanism is active transport of ribonucleoprotein (RNP) complexes containing specific mRNAs, requiring motor proteins and cytoskeletal elements. This is used by Bicoid and Oskar mRNAs in the Drosophila oocyte (Lane and Kalderon, 1994; Wilsch-Brauninger et al., 1997), MBP mRNA in Oligodendrocytes (Ainger et al., 1993), ASH1 mRNA in budding yeast (Bertrand et al., 1998), ß-actin mRNA to the axons of neurons (Morris and Hollenbeck, 1995) and mRNAs transported via the late pathway in Xenopus oocytes (Melton, 1987).
1.4.1. Diffusion and entrapment
The initial accumulation of early localized mRNA to the Balbiani body of Xenopus oocytes (illustrated in Fig. 1.7) has been shown not to require intact microtubules or microfilaments (Kloc et al., 1996). However, the mode of migration by which the Balbiani body fragments together with the associated mRNAs reach the vegetal cortex is still not known. Recent studies have shown that labelled Xcat2 and Xdazl mRNA particles move randomly throughout the cytoplasm of stage I oocytes and continuously aggregate within the METRO substructure of the Balbiani body in a manner distinguishable from the non-localized ß-globin mRNA (Chang et al., 2004). Drosophila Nanos which is related to Xcat2 is also localized by the same mechanism, however, the factors that mediate the entrapment of Xcat2 in Xenopus oocytes remain to be identified. The cis-acting localization elements (LE), usually residing within the 3´UTR, have been mapped for several early localized mRNAs, e.g. Xcat2 mRNA contains in the 3´UTR a 127 nt long
mitochondrial cloud LE (Zhou and King, 1996) and a 164 nt germinal granule LE (Kloc et al., 2000).
Figure 1.7 Model of the mechanism of diffusion and entrapment used by early localized mRNAs in oocytes.
During stage I of oogenesis, mRNAs (red dots) become selectively anchored within the Balbiani body, which attaches to the nucleus (blue) and contains mitochondria (green) and ER membranes.
In stage II, the mRNAs co-migrate with fragmented Balbiani body to the vegetal cortex, [from (Kloc et al., 2002a)].
1.4.2. Active transport
Transport of late localizing mRNAs, such as Vg1 mRNA, to the vegetal cortex is dependent on intact microtubules and the anchoring step relies on intact microfilaments (Yisraeli et al., 1990). This finding was the first indication that the late pathway operates by the active directional transport mechanism, and later kinesin I (Yoon and Mowry, 2004) as well as heterotrimeric kinesin (kinesin II) (Betley et al., 2004) have been proposed to be the motor proteins that mediate this process.
Since kinesins are plus directed, the observation that the majority of microtubules in stage III oocyte have their minus end directed towards the vegetal cortex (Pfeiffer and Gard, 1999), could mean that there exist unique tracks of microtubules in stage II-IV oocytes which have their plus end oriented towards the vegetal cortex. These distinct microtubule tracks have been proposed to be established by a microtubule organising center consisting of a γ-tubulin positive centrosome present in the Balbiani body at stage I (Kloc and Etkin, 1998). The preceding accumulation of the mRNAs within the wedge-shaped structure during
late stage II takes place in a microtubule-independent manner (Kloc and Etkin, 1998). The active transport process in oocytes is thought to proceed as illustrated in Fig 1.8; several trans-acting factors have been identified as playing a role in this process. The mRNAs have cis-acting vegetal localization elements (LE) usually residing in the 3´UTR, which have been shown to be necessary and sufficient to assemble the transport RNP complex. There are four major steps in this process;
firstly, there is nuclear assembly of the core RNP complex involving the recruitment of VgRBP60/PTB along with its direct interacting partner Vg1RBP to the LE which is then exported into the cytoplasm (Kress et al., 2004). In Drosophila oocytes, the localization of Oskar mRNA has been shown to be coupled with nuclear splicing of the pre-mRNA, since mRNA from an intronless Oskar gene is unable to localize to the posterior pole (Hachet and Ephrussi, 2004).
Secondly, when the RNP complex reaches the cytoplasm it undergoes extensive remodelling; VgRBP60 which in the nucleus recruits Vg1RBP to the LE mediates the remodelling of the Vg1RBP to interact directly with the RNA (Lewis et al., 2007). The RNP maturation results from the recruitment of several additional factors such as XStaufen1 (Allison et al., 2004; Yoon and Mowry, 2004), Prrp (Zhao et al., 2001) and presumably 40LoVe (Czaplinski et al., 2005; Czaplinski and Mattaj, 2006). 40LoVe may well associate with the LE in the nucleus given its strong nuclear accumulation (Czaplinski and Mattaj, 2006).
The third step involves the coupling of the transport competent RNP complex to the motor proteins (i.e. kinesins); it is currently not known how this step is achieved, although the kinesins involved have been described (as mentioned above). The final step of anchoring has been shown to require cytokeratin filaments (Kloc et al., 2005), a function which had previously been linked to microfilaments (Yisraeli et al., 1990). The reported delocalization of Vg1 mRNA upon application of agents disrupting the microfilaments could thus be due to requirement of F-actin for linking the cytokeratin network to the vegetal cortex (Gard et al., 1997). Interestingly, the localized RNAs of VegT and Xlsirts have been reported to be integral to the network of cytokeratin (as illustrated in Fig 1.8), mediating the anchoring of the majority of the vegetally localized mRNA (Kloc et
al., 2005). Protein components of the transport complex appear to remain largely anchored with the mRNA; immuno-florescence studies have shown that these proteins share a vegetal cortex enrichment pattern reminiscent of the localized mRNAs (Czaplinski et al., 2005; Kress et al., 2004; Yoon and Mowry, 2004; Zhang et al., 1999).
Figure 1.8 Model of the active transport mechanism used by mRNAs localizing via the late pathway
Based on (Jansen, 2001). 1: Formation of core localizing RNP complex in the nucleus, Vg1RBP associates with the LE through VgRBP60 (Kress et al., 2004) 2: mRNA export into the cytoplasm.
3: Remodelling of the RNP through the activity of VgRBP60, Vg1RBP binds directly to the LE (Lewis et al., 2007); additional factors such as XStaufen1, Prrp and 40LoVe are recruited to form the mature, transport competent RNP complex (size not drawn to scale). 4: Coupling of the mature RNP complex to kinesin and transport along microtubules to deliver the cargo to the vegetal cortex. 5: Anchoring of the mRNA to the cytokeratin network, which involves VegT and Xlsirts RNAs; illustration was taken from (Kloc et al., 2005). The illustration of microtubules and kinesin is from (Cooper, 2000).
1.4.3. Cis-acting elements
mRNAs destined for vegetal localization usually harbour sequence elements that have been experimentally determined to be necessary and sufficient to mediate the process in their 3´ UTR; however, XNIF mRNA has been reported to have the localization element (LE) residing in the 5´UTR (Claussen et al., 2004). Vegetal localization elements mapped so far do not appear to share conserved primary sequences (King et al., 2005), whilst secondary structures have not yet been described for these LEs. The LEs vary in size, they can be as long as 340 nt for Vg1 LE (Mowry and Melton, 1992) and 300 nt for VegT (Bubunenko et al., 2002;
Kwon et al., 2002). The shortest LE of only 25 nt that mediates localization in oocytes has been mapped for fatvg mRNA (Chan et al., 1999).
Multiple short redundant sequence elements have been found to cluster in the LEs of Vg1 and VegT; these are the so-called E2 (A/CYCAC) and VM1 (YYUCU) sequence elements (Kwon et al., 2002; Lewis et al., 2004). The E2 elements have been proposed to serve as a binding site for Vg1RBP (Deshler et al., 1998) and VM1 may recruit VgRBP60 to the LE (Cote et al., 1999; Lewis et al., 2004).
Mutational analysis showed that these elements are crucial for the localization of both Vg1 and VegT mRNAs. However, the non-existence of E2/VM1 clusters in the LEs of Xvelo1, XNIF and XDead end mRNA (Claussen et al., 2004; Claussen and Pieler, 2004; Horvay et al., 2006), argues that they are not absolutely required for vegetal localization. Similarly, competition experiments using multimerized E2 or VM1 elements revealed that whilst molar excess E2 element inhibited the localization of Vg1 LE that of VM1 rather improved the localization efficiency of Vg1 LE (Czaplinski and Mattaj, 2006), suggesting that the VM1 interacting proteins perhaps different from VgRBP60 may function as inhibitors of localization.
Figure 1.9 Structure of mRNA depicting its vegetal localization element in the 3´ UTR.
1.4.4. Trans-acting factors
Vegetal mRNA localization is achieved by the specific recruitment of trans-acting factors to the LE. How these factors work together to exert their function is described above. In Xenopus oocytes factors have been identified by means of biochemical approaches, unlike the predominantly genetic approach used in Drosophila. UV crosslinking analyses have identified a set of proteins, (p78, p69, p60, 40, p36, p30) as binding specifically to Vg1 LE (Cote et al., 1999). The majority of trans-acting factors identified to date in Xenopus oocytes and in other systems has turned out to be RNA binding proteins with classical RNA binding domains. Interestingly, the endosomal sorting complex has been shown to play a role in Bicoid mRNA localization in Drosophila oocytes, and its component proteins exhibit a conserved sequence specific RNA binding activity (Irion and St Johnston, 2007).
1.4.4.1. Vg1 mRNA binding protein - Vg1RBP
Vg1RBP is an RNA binding protein of about 69 kDa that contains two RRM and four KH domains as depicted in Fig. 1.10; it was independently identified twice as trans-acting factor of vegetal mRNA localization in Xenopus oocytes, firstly as protein binding specifically to the Vg1 LE (Yaniv and Yisraeli, 2001) and secondly as co-fractionating with ER membranes and mediating the association of Vg1 mRNA with an ER sub-compartment to promote its vegetal localization (Deshler et al., 1998; Deshler et al., 1997). All four KH domains are required for Vg1 LE binding, whilst the KH domains 3 and 4 mediate homodimerization (Git and Standart, 2002). Vg1RBP mediates the association of Vg1 mRNA to microtubules in vitro (Elisha et al., 1995; Havin et al., 1998), an RNA binding mutant version of Vg1RBP still localizes to the vegetal cortex, suggesting that the process is dependent on its microtubule interaction and not on RNA binding per se (Rand and Yisraeli, 2007).
Figure 1.10 Domain structure of Xenopus Vg1RBP protein
Figure 1.10 Domain structure of Xenopus Vg1RBP protein, showing 2 RRM domains in the N- terminal and 4 KH domains in the C-terminal portion, drawn to the scale as predicted by the NCBI- protein blast program.
A homolog of Vg1RBP, ZBP1, has a function in ß-actin mRNA localization to the leading edge of fibroblast cells, dendritic filopodia and filopodial synapses in neurons (Eom et al., 2003; Ross et al., 1997). Additionally, it has a Src kinase mediated function of activation of translation upon its mRNA cargo reaching the periphery of the cell (Huttelmaier et al., 2005). It also plays a role in the regulation of mRNA stability in response to cellular stress in U2OS cells (Stohr et al., 2006).
In Drosophila oocytes, insulin-like growth factor II mRNA–binding protein 1 (IMP1), also a homolog of Vg1RBP, is linked to gurken expression (Geng and Macdonald, 2006) and colocalizes with Oskar mRNA at the posterior pole;
surprisingly only the mutation of IMP1 binding site in the 3´UTR affected the translation and anchoring of Oskar mRNA at the posterior, but a IMP1 protein null mutation was found not to have any such effect (Munro et al., 2006).
1.4.4.2. XStaufen1 protein
XStaufen1 is a double-stranded RNA binding protein, 79 kDa in size and consisting of 5 DSRBD and a tubulin binding domain as illustrated in Fig 1.11.
Staufen was first isolated in Drosophila, as a gene required for anterior-posterior patterning (Schupbach and Wieschaus, 1986), and subsequently shown to be involved in the localization of both Bicoid mRNA to the anterior and Oskar mRNA to the posterior poles of the oocyte (St Johnston et al., 1991, Ferrandon, 1994
#246). Xenopus Staufen was recently cloned and two protein isoforms were found to be expressed in the oocyte (Allison et al., 2004); XStaufen1 is the predominantly expressed isoform and plays a role in the vegetal mRNA localization. It interacts with Vg1 and VegT mRNAs and a dominant negative form which is able to inhibit localization also perturbs the interaction between Vg1RBP and VgRBP60 (Yoon and Mowry, 2004). In rat neurons, Staufen proteins have been identified as components of large RNA particles and are required for dendritic mRNA localization (Kiebler et al., 1999).
Figure 1.11 Domain structure of Xenopus Staufen 1 protein
It has 5 DSRB domains and a tubulin binding domain, drawn to the scale as predicted by the NCBI-protein blast program.
Drosophila Staufen exhibits distinct roles using two of its different conserved domains (illustrated in Fig. 1.12); the inserted loop region of DSRB domain 2 is required for posterior pole localization of Oskar mRNA, whilst the DSRB domain 5 is implicated in de-repression of localized Oskar mRNA in oocytes (Micklem et al., 2000). Interestingly, the interaction with a protein called Miranda can switch localization of Oskar mRNA from the posterior pole to the anterior pole (Schuldt et al., 1998).
Figure 1.12 Model for the function of the Drosophila Staufen protein
[Taken from (Schuldt et al., 1998)], showing the association of the protein to Oskar mRNA; the DSRB 2 has an inserted loop region.
1.4.4.3. VgRBP60/PTB protein
VgRBP60 is the Xenopus homolog of human hnRNP I; it is approximately 60 kDa in size and contains four RRM domains (Fig. 1.13); it is also referred to as polypyrimidine track binding protein (PTB) with a sequence-specific binding activity to CU-rich motifs and implicated in the regulation of splicing (Coutinho- Mansfield et al., 2007). VgRBP60 was isolated as a protein specifically binding to the VM1 repeats in the Vg1 LE and mutations in this binding site blocked vegetal mRNA localization in oocytes (Cote et al., 1999). Interestingly, VgRBP60 recruits
Vg1RBP to the LE in the nucleus (Kress et al., 2004) and mediates the remodelling of the transport complex upon export into the cytoplasm leading to a switch of indirect association to direct interaction with the LE (Lewis et al., 2007).
Figure 1.13 Domain structure of Xenopus VgRBP60 protein, depicting the 4 RRM domains
1.4.4.4. Xenopus 40LoVe protein
40LoVe was identified as a 40 kDa vegetal localization protein, which binds specifically to the LE of VegT and Vg1 mRNA; it contains of two RRM domains (Fig 1.14) and it is a member of the hnRNP D family of proteins; three protein isoforms are detected in the oocyte (Czaplinski et al., 2005). It interacts with Vg1RBP and VgRBP60 in an RNA dependent manner and also exhibits a vegetal cortex enrichment reminiscent of the localization pattern of its target mRNA (Czaplinski and Mattaj, 2006). Other hnRNP D proteins, e.g. AUF1, have been implicated in the regulation of mRNA stability, their binding to AU rich elements (ARE) elicits rapid degradation of target mRNAs (Sarkar et al., 2003).
Figure 1.14 Domain structure of Xenopus 40LoVe (Localization Vegetal) protein depicting the two RRM domains.
1.4.4.5. Xenopus Proline-rich RNA binding protein - Prrp
Prrp is an RNA binding protein approximately 40 kDa in size and made up of two RRM domains (Fig 1.15) and a proline-rich domain in the C-terminal region, which directly interacts with Profillin. Xenopus Prrp was isolated through a screen of a phage-cDNA library for Vg1 LE binding proteins; it interacts with endogenous late localizing mRNAs but not early localized ones. Additionally, it co-localizes with Vg1 mRNA at the vegetal cortex (Zhao et al., 2001).
Figure 1.15 Domain structure of Xenopus Prrp protein depicting the 2 N-terminal RRM domains and a C-terminal Proline-rich domain
1.4.4.6. VgRBP71 protein
In the same approach used for isolating Xenopus Prrp, another Vg1 LE interacting protein was identified, called Xenopus VgRBP71. It is about 71 kDa in size and contains four KH domains (Fig. 1.16) (Kroll et al., 2002). VgRBP71 is the Xenopus homolog of human KSRP, implicated in the regulation of splicing (Min et al., 1997). Apart from Vg1 mRNA, VgRBP71 also associates with other localized mRNAs such as for VegT and Xcat2 and is involved in protein-protein interactions with Prrp. VgRBP71 has been proposed to mediate the cleavage of the Vg1 mRNA eliminating the translational control element and thus leading to de- repression of translation during late oogenesis (Kolev and Huber, 2003). Although there is no evidence that VgRBP71 has a role in vegetal mRNA localization, ZBP2 the Chicken homolog, has been found to play a role in ß-actin mRNA localization in fibroblasts and neurons, since a truncated version is able to inhibit this process (Gu et al., 2002).
Figure 1.16 Domain structure of Xenopus VgRBP71 protein, showing 4 KH domains drawn to the scale as predicted by the NCBI-protein blast program
1.5. Transport RNP: size, composition and structure
Several mRNA molecules which localize have been observed to form a large transport particle, or mRNA granules, needed to achieve localization in a number of systems and termed “locusome” (Bassell et al., 1999) or “locasome” (Gu et al.,
2004) in analogy to ribosomes or spliceosomes. Myelin basic protein (MBP) mRNA granules have been observed in oligodendrocytes and were estimated to have a radius between 0.6 and 0.8 µm, they colocalize with arginyl-tRNA synthetase, elongation factor 1alpha and rRNA (Barbarese et al., 1995). In Drosophila oocytes, Bicoid mRNA forms Staufen containing particles, held together in part by RNA-RNA interactions (Ferrandon et al., 1994); a fusion protein of GFP-Exuperantia has also been shown to form particles that migrate through ring canals into oocytes (Wang and Hazelrigg, 1994). mRNA transport particles could be biochemically isolated, e.g. the ASH1 mRNA locasome was isolated using affinity tag She2p and found to contain several additional proteins (Gu et al., 2004). Similarly, GFP-Exuperantia particles which migrate in high density fractions could also be isolated with an antibody against GFP and found to contain Yps protein and Oskar mRNA (Wilhelm et al., 2000a). In human cells (HEK cells), insulin-like growth factor II mRNA–binding protein (IMP) granules were estimated to be 100-300 nm in diameter and to consist of IMP proteins, 40S ribosomal subunits, shuttling hnRNPs, poly(A)-binding proteins, and several mRNAs (Jonson et al., 2007).
In Xenopus oocytes, Xcat2 RNA was observed in particles that move randomly throughout the cytoplasm (Chang et al., 2004), whilst XStaufen1 protein have been observed in a high density RNP in gradient centrifugation separation of oocyte lysate (Yoon and Mowry, 2004), and as large particle on size exclusion columns (Allison et al., 2004).
These transport mRNA granules are thought to reflect structures that are needed to accomplish the process of mRNA localization; the large size, in some cases many times larger than ribosomes, could be accounted for by the many proteins, mRNAs and sometimes also translational machinery that they contain (Jansen, 2001).
1.6. Translational control of localized mRNAs
The widely held belief regarding the relationship between mRNA localization and translational control is that during transport, translation must be repressed to allow for the subsequent local protein synthesis. There are several ways in which
localized mRNA are controlled translationally in the various biological systems where this phenomenon occurs.
1.6.1. Drosophila oocytes/embryos
A number of mRNAs are know to be localized during oogenesis, Nanos mRNA is translated only upon reaching the posterior pole, so unlocalized Nanos is repressed in Drosophila embryos through the activity of a protein called smaug (Smibert et al., 1999; Smibert et al., 1996), which requires a 90 nt translational control element in its 3´UTR that is distinct from the localization element (Gavis et al., 1996). Oskar mRNA localization is also coupled to its translation; it is repressed by a number of proteins to ensure that no translation occurs apart from at the posterior pole, reflecting its key role as a posterior determinant. Repression of Oskar mRNA is achieved by proteins such as Bruno through the Bruno response element in the 3´UTR (Castagnetti et al., 2000; Chekulaeva et al., 2006;
Gunkel et al., 1998) and also by Hrp48 through its association with both 5´ and 3´UTRs (Huynh et al., 2004; Yano et al., 2004).
1.6.2. Neurons and Fibroblast cells
mRNA encoding Myelin basic protein (MBP) is repressed during transport to the dendrites in oligodendrocytes by the recruitment of hnRNP E1 to the mRNA by hnRNP A2, which binds to its response element (A2RE) that resides in the 3´UTR (Kosturko et al., 2006). ß-actin mRNA is repressed in a similar manner during transport to the axons of neurons and to the leading edge of migrating fibroblasts by ZBP1, dependent on the 54 nt zipcode in the 3´UTR, the same element that mediates the localization process; translation is activated at the destination by the activity of Src kinase on ZBP1 (Huttelmaier et al., 2005).
1.6.3. Xenopus oocytes
The only example of translational control elements (TCE) described for localized mRNAs in Xenopus oocytes is for Vg1 mRNA. Two overlapping Vg1 mRNA TCEs have been mapped in the 3´UTR; they are distinct from the LE and ElrB protein has been implicated in mediating the repression during early oogenesis by direct
binding to the 250 nt Vg1 TCE (Colegrove-Otero et al., 2005), whilst a 38 kDa unknown protein was found to bind the 350 nt TCE and also suggested to mediate repression (Wilhelm et al., 2000b).
1.7. Aims of the project
mRNA localization in Xenopus oocyte is thought to occur by specific recruitment of trans-acting factors to the localization element; several of these factors have been identified, including Vg1RBP, VgRBP60 and XStaufen1. However, UV crosslinking analysis using LEs reveals proteins that do not correspond to the factors identified so far (Horvay et al., 2006). Identification of the complete set of proteins interacting with a localized mRNA is imperative since this will make it possible to improve the understanding of the mechanisms involved. The first part of the project is an attempt to identify novel trans-acting factors with a function in vegetal mRNA localization; for this, a biochemical purification strategy is established. This strategy was based on the principle that localized mRNAs and the associated trans-acting factors form large RNP complexes. The second part involved the functional characterization of candidate proteins identified with a focus on (i) their interaction with known localization proteins, (ii) their subcellular distribution, and (iii) their role in the process of localization of mRNAs to the vegetal cortex of oocytes, as well as (iv) for possible function in other aspects of mRNA metabolism.
2. Materials and Methods 2.1.1. Model Organism
Oocytes and embryos used in this work were from African clawed frogs Xenopus leavis (family: Pipidae). Pigmented and albino frogs were ordered from Nasco (Fort Atkinson, Wisconsin, USA) and Dipl.-Ing. Hoest Kähler (Hamburg, Germany),
2.1.2. Bacteria strains
Escherichia coli (E. coli) strains used were obtained from Stratagene XL1-Blue: RecA1, endA1, gyrA96, thi-1, hsdR17, supE44, relA1, lac[F´proAB, lacIqZΔM15, Tn10(Tetr)]c.
BL21 (DE3): E.coli B F-, ompT, hsdS(r
B- m
B-), dcm+, Tetr, gal λ(DE3)
endA Hte [argU proLCamr] [argU ileY leuW Strep/Specr]
2.1.3. List of Chemicals
5-Bromo-4-chloro-indoxyl-β-D-galactoside (X-Gal) Roth Acrylamide Roth Agarose Roth Ampicillin Roche Bromophenol blue Sigma Blendzyme (Collagenase) Roche CHAPS Sigma Chloroform Merck Coomassie Brilliant Blue G-250 Serva Brilliant Blue R-250 Serva
Digoxigenin UTP Sigma Dimethyl Sulfoxide (DMSO) Sigma
Dithiothreitol (DTT) Biomol Ethidiumbromide Sigma
Ethylenediaminetetraacetic acid (EDTA) Roth Formaldehyde Merck
Glycerol Merck
